Our goal is to develop effective cationic liposome:DNA complex (CLDC)-based systemic gene therapy for metastatic human breast cancer. Tumor metastasis is the major cause of cancer death. Thus, meaningful reductions in cancer mortality require effective, systemic therapy. Our primary hypothesis is that new, more efficient cationic liposomes and expression vectors have rendered CLDC-based intravenous (iv) gene delivery both an effective anti-metastatic therapy, and a powerful tool for elucidating the regulation of tumor angiogenesis. We have already shown that CLDC-based iv gene delivery can i) significantly reduce both metastatic spread and tumor angiogenesis in tumor-bearing mice, ii) transfect very large numbers of metastatic tumor cells, endothelial cells and macrophages, iii) maintain therapeutic levels of gene expression for greater than or equal to 2 months, and iv) then efficiently re-express the gene following repeated re-injection in immunocompetent mice. We will test this iv CLDC gene delivery approach in two metastatic tumor models: C3H/HeN mice bearing the syngeneic mouse mammary carcinoma line, C3L5 and in SCID/beige mice bearing the human breast cancer xenograft line, MDA-MB-435. Our five specific aims are: Aim 1: Determine the anti-metastatic, anti-angiogenic and pro-apoptotic effects produced by a variety of anti-tumor genes, following their CLDC-based iv gene delivery into tumor-bearing mice. Aim 2: Increase anti-tumor activity by identifying CLDC co-delivered genes that produce synergistic anti-cancer activity in mice. Aim 3: Use our novel, durably- expressing EBV-based plasmid vector to attempt to: 3A: Produce sustained eradication of metastatic breast cancer, and 3B: Provide long-term anti-metastatic protection in mice bearing localized breast tumors. AIM 4: Increase therapeutic activity and reduce host toxicity by using two cell type-specific promoters to target the expression of CLDC-delivered genes: 4A: A 900 bp sequence of the Von Willibrand Factor (VWF)-3 promoter element incorporated into our expression plasmid to specifically target expression of CLDC delivered anti-angiogenic genes to vascular endothelial cells. 4B: A 694 bp sequence of the human mucin-like glycoprotein promoter DF3 incorporated into our expression plasmid to target expression of iv, CLDC-delivered genes to breast cancer cells that overexpress DF3. Aim 5: Use both combination gene delivery and promoter-based targeting studies to better characterize the genetic and cellular factors that regulate the angiogenic phenotype in tumor-bearing mice. These studies should both improve system anti-cancer gene therapy and identify specific genes and cell types that can control tumor angiogenesis in tumor-bearing animals.